Viruses, especially the hyper-mutable retroviruses such as HIV, can rapidly become resistant to drugs used to treat the infection. This extreme mutability has allowed HIV to diverge into two major species, HIV-1 and HIV-2, each of which has many types, subtypes and drug-resistant variations.
Strategies for coping with the emergence of viral drug-resistant strains include combination drug therapies (see, e.g., Lange (1996) AIDS 10 Supplement 1:S27-S30). Drugs against different viral proteins and drugs against multiple sites on the same protein are commonly used as a strategy to overcome the adaptability of the virus. Combination therapies for retroviruses, using, e.g. protease inhibitors and nucleoside analogues, such as AZT, ddI, ddC and d4T, can become ineffectual. The virus can develop a complete resistance to the drugs in a relatively short period of time (see, e.g., Birch (1998) AIDS 12:680-681; Roberts (1998) AIDS 12:453-460; Yang (1997) Leukemia 11 Supplement 3:89-92; Demeter (1997) J. Acquir. Immune Defic. Syndr. Hum. Retrovirol. 14(2):136-144; Kuritzkes (1996) AIDS 10 Supplement 5:S27-S31). Furthermore, although recent advances in testing HIV-SIV vaccine strategies in monkeys have been reported (see, e.g., Amara et al, (2001) Science 291:1879-1881), no antiretroviral vaccine with proven effectiveness is currently available for use in humans (see, e.g., Haynes (2001) Immunol. Res. 22:263-269; Bolognesi (1998) Nature 391:638-639; Bangham (1997) Lancet 350:1617-1621).
The HIV-1-caused AIDS epidemic began about 20 years ago. As of December 2000, the total number of cases of HIV infection and AIDS since the start of the epidemic was estimated to be around 58 million worldwide, which includes 22 million deaths and 36 million people living with HIV/AIDS (UNAIDS-WHO Internet report). Thus, there exists a great need for compounds that are more effective against retroviruses such as HIV-1, especially against varieties of HIV that have developed resistance to current treatments.
A “zinc finger” motif is a highly conserved and essential structure found in many viruses, including retroviruses such as HIV. For example, Gag and Gag-Pol proteins in the Retroviridae, except for Spumaviruses, contain a highly conserved zinc finger motif (Cysteine-Cysteine-Histidine-Cysteine: CCHC) within the nucleocapsid p7 (NCp7) protein portion of the polyprotein (see definitions below). Mutations of the chelating residues in the zinc fingers yield a non-infectious virus. The absolute conservation of the metal chelating cysteine and histidine residues along with other residues of the protein, and the participation of NCp7 in essential functions during early and late phases of virus replication, identifies this feature as a particularly appealing antiviral target for hyper-mutable retroviruses, including HIV. Because these zinc fingers are identical in most retroviruses, drugs targeting zinc fingers could provide broad spectrum antivirals that would greatly reduce resistance issues.
Various C-nitroso compounds, disulfide-containing compounds and azoic compounds, such as cystamine, thiamine disulfide, disulfiram and azodicarbonamide (ADA) can oxidize zinc finger cysteine thiolates and induce intra- and inter-molecular disulfide cross-linking (see, e.g., McDonnell (1997) J. Med. Chem. 40:1969-1976; Rice (1997) Nature Medicine 3:341-345; Rice (1997) Antimicrob. Agents and Chemotherapy 41: 419-426; Rice (1996) J. Med. Chem. 39:3606-3616; Rice (1996) Science 270:1194-1197; Rice (1993) Proc. Natl. Acad. Sci. USA 90:9721-9724; Rice (1993) Nature 361:473-475; Henderson, et al. WO 96/09406; Vandevelde (1996) AIDS Res. Hum. Retroviruses 12:567-568). Cysteine thiols in each of the two zinc fingers of HIV are rapidly attacked by reagents such as Cu2+, Fe3+, C-nitroso compounds, disulfides maleimides, alpha-halogenated ketones and nitric oxide derivatives, with simultaneous loss of the native protein structure. For example, treatment of intact HIV-1 with an oxidizing agent, such as 3-nitrosobenzamide, a C-nitroso compound, induces disulfide linkage of the nucleocapsid protein and inactivates viral infectivity through oxidation of the zinc fingers (Rice (1993) Nature 361:473; Rice (1993) Proc. Natl. Acad. Sci. USA 90:9721-9724). C-nitroso compounds can also inactivate eukaryotic CCHC zinc finger containing poly(ADP-ribose) polymerase (Buki (1991) FEBS Letters 290:181). However, these compounds tend to be toxic, have poor solubility and bioavailability, and are quickly reduced and inactivated in biological solutions.
It has recently been shown that certain compounds containing acylthio groups interact with the NCp7 zinc fingers via their acylthiol moiety. This interaction involves an acyl transfer from the acylthiol to a target cysteine sulfur atom and does not require an oxidation step as with the above-mentioned compounds (Turpin et al. (1999) J. Med. Chem. 42:67-86; Basrur et al. (2000) J. Biol. Chem. 275:14890-14897). Various members of this class of acylthiol compounds frequently exhibited acceptable antiviral potency. Pyridinioalkanoyl thioesters (PATEs) exhibited superior anti-HIV-1 activity with minimal cellular toxicity and showed appreciable water solubility. PATEs were shown to preferentially target the NCp7 C-terminal zinc finger when tested against other molecular targets. These compounds thus possessed broad spectrum antiviral activity, particularly against retroviruses such as HIV.
Despite the promising activity of the PATE compounds, the compounds of the present invention provide a significant advance in the treatment of viral infections. The PATE compounds are charged species, and as such they are quite hygroscopic. This makes isolation and purification difficult. More importantly, it increases hydrophilicity so much that uptake is inhibited and excretion is accelerated. As a result, those compounds have poor delivery characteristics for oral administration. Furthermore, many compounds of the present invention show improved hydrolytic stability in test systems containing serum, which are used to estimate the in vivo lifetime of compounds to be evaluated as drug candidates.
The compounds of the present invention possess broad spectrum antiviral activity like the PATEs. However, the acylthiols and thiols of the present invention are generally neutral species at physiological pH, so they provide better delivery characteristics than the PATE compounds. The acylthiol compounds of the present invention have increased in vivo stability, which allows more efficient delivery of the active acylthiol species to the targeted zinc finger viral proteins. Compounds of the present invention also possess other advantages, such as greater in vivo stability and greater ease of synthesis, isolation, purification, and storage, which make them still more advantageous relative to the PATEs. The compounds of the present invention are thus useful for the prevention and treatment of retroviral infections, for the preparation of inactivated viruses, and for the detection and production of antibodies to inactivated viral proteins.
The compounds of the present invention, by virtue of their antiviral activity, are also useful for the manufacture of compositions that can be used to inactivate viruses in vitro or in vivo. These compositions can be used to inactivate viruses to prevent the transmission of a viral infection, to treat a viral infection, or to prepare an inactivated virus as, for example, to prepare a vaccine. They are especially useful for the manufacture of medicaments that can be used to treat mammals infected with viral diseases, particularly retroviral diseases such as, for example, HIV.